Registration Dossier

Administrative data

Description of key information

The skin irritation potential was estimated using Danish QSAR Models. Using this model the skin irritation potential of the test item was estimated to be not skin irritating.

Studies will be performed in accordance with OECD TG 439 and OECD TG 431 in order to determine the skin irritation/corrosion potential of the test item. Furthermore, in order to determine the eye irritation potential of the test item, studies will be performed according OECD TG 492 and OECD TG 438. The studies were initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04 March 2020 to 05 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
14 June 2019
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.
Version / remarks:
20 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” (ECVAM Database Service on Alternative Methods to Animal Experimentation).
Version / remarks:
updated December 2011 / February 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approx. 25 μL, one time
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL per well
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (Cat. 1A) if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive to skin (Cat 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% AND after 60 minutes exposure smaller than 35%; or if the viability after 60 minutes exposure is greater than or equal to 35% AND after 240 minutes exposure smaller than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.
- Justification for the selection of the cut-off point: The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998). The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2015).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
4 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks on result:
other: results not yet available
Conclusions:
The skin corrosion potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 431 in order to determine the skin corrosion potential of the test item. The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8
Version / remarks:
February 2009
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult human donors
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. It showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin(TM)SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution; rest of the PBS was removed from epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 µL

VEHICLE
- Amount(s) applied: 10 μL

POSITIVE CONTROL
- Amount(s) applied: 10 μL
- Concentration: 5% aq. solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks on result:
other: results not yet available
Conclusions:
The skin irritation potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 439 in order to determine the skin irriation potential of the test item. The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
GLP compliance:
yes (incl. certificate)
Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3ºC to 20.4 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Time interval prior to initiating testing: 2 h
- indication of any existing defects or lesions in ocular tissue samples: After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: The test item was applied in such a way that the approximately 2 cm^2 entire surface of a plastic film was covered with test substance.
Duration of treatment / exposure:
10 sec
Duration of post- treatment incubation (in vitro):
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75,120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity.Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye. The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 or 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and, after being placed in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluoresce in retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl (9 g/L saline)

POSITIVE CONTROL USED: Acetic acid 10 % (v/v)

APPLICATION DOSE AND EXPOSURE TIME: 2 cm^2 for 10 sec

OBSERVATION PERIOD: up to 240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored.After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: decision criteria as indicated in the TG was used.
Irritation parameter:
percent corneal swelling
Remarks on result:
other: results not yet available
Irritation parameter:
cornea opacity score
Remarks on result:
other: results not yet available
Irritation parameter:
fluorescein retention score
Remarks on result:
other: results not yet available
Conclusions:
The eye irritation potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 438 in order to determine the eye irriation potential of the test item. The results will be updated as soon as available.The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritati on of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
29 June 2015
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular™ (OCL-200-EIT), Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma, resulting in "not detected". The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3 % (v/v) Triton X-100).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours at 37 ± 1 °C
Duration of post- treatment incubation (in vitro):
Post-Soak: 25 minute at room temperature
Post-treatment Incubation: 18 hours at 37 ± 1 °C
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT), Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used: see above
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: see above
- Description of any modifications to the test procedure: None
- Number of tissue replicates used per test chemical and controls: see above
- Wavelength used for quantifying MTT formazan, and measuring device: 96-well plate spectrophotometer at the wavelength of 570 nm
- Description of the method used to quantify MTT formazan: Absorbance / Optical Density of the samples
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 % of the negative control. The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

- Acceptable variability between tissue replicates for positive and negative controls: The mean OD value of the two negative control tissues should be between 0.8 and 2.5. The acceptable percentage viability for positive control (mean of two tissues) is for 30 minute exposure below 50 % of control viability and for 6 hours exposure below 50 % of control viability.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single chemical is < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals).
Irritation parameter:
other: tissue viability [%]
Remarks on result:
other: results not yet available
Conclusions:
The eye irritation potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 492 in order to determine the eye irriation potential of the test item. The results will be updated as soon as available.The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Additional information

Justification for classification or non-classification